A Novel miRNA Detection Method Using Loop-Mediated Isothermal Amplification

A novel ligation-based loop-mediated isothermal amplification has been developed for miRNA detection. Two stem-loop structure DNA linker A/B probes which hybridized with miRNA were designed to establish a rapid and ultrasensitive miRNA-LAMP system for miRNA detection. Target miR-200a was used to template the ligation of Linker A/B probes with SplintR Ligase and used as a dumbbell-shaped amplicon. By adding BIP/FIP and Bst 2.0 DNA polymerase, the LAMP reaction was carried out, which brought greatly improved amplification efficiency. The double-stranded DNA fluorescent dye EvaGreen was added for the detection of amplification product to achieve the quantification of the target miRNA. This method can detect miRNA in a linear range of seven orders of magnitude, with a detection limit of 100 fM. Therefore, this ultrasensitive miRNA-LAMP assay provides a new path for the highly sensitive quantitative analysis of miRNA, thereby bringing convenience to clinical diagnosis and prognostic research.


Introduction
MicroRNAs (miRNAs) comprise a family of small singlestranded non-protein-coding RNAs (20-25 nucleotides) that have recently emerged as post-transcriptional regulators of gene expression [1,2].In addition, miRNAs play critical roles in various biological processes and genetic pathways.Tey are also involved in a number of human diseases including cancer [3][4][5][6], which have been hypothesized to play an important role in the control of tumor growth and eventually lead to more roles and progression [7].miRNAs have been regarded as promising biomarkers for clinical diagnosis and treatment [8,9].However, miRNA detection is very challenging because of the intrinsic characteristics of these molecules, such as small size, sequence similarity among various members, and lack of common features.
Currently, more than 2000 miRNAs have been annotated in humans (miRBase: http://www.mirbase.org/index.shtml), and computational predictions indicate that miRNAs may regulate the expression of 60% of all human protein-coding genes [10].MiRNAs play a pivotal role in critical biological processes, including cellular growth, proliferation, and diferentiation [11,12].A growing body of evidence has demonstrated the importance of miRNAs in managing chemotherapy efcacy in multiple human cancers, and they might also function as tumor suppressors and oncogenes.miRNAs have opened a new window to an important area of biology that was previously unexplored and also have important implications in human development and diseases.Tus, the rapid and sensitive quantifcation of miRNAs is of great importance to the understanding of their biological functions and clinical applications.
LAMP is an isothermal method that was frst described by Notomi et al. [35].In a typical LAMP reaction, there are at least four primers: two inner and two outer primers.Tese primers were designed to recognize a set of six distinct sequences, displaced by the on the autocycling strand displacement activity DNA polymerase and generates a large amount of amplifed products within 1 h; moreover, the method can be simplifed by running at a constant temperature, eliminating the need for temperature cycling during amplifcation [34].Te mechanism of LAMP amplifcation reaction includes three steps: production of starting material, cycling amplifcation and elongation, and autocycling strand displacement.Terefore, the limit of detection and dynamic range of the nucleic acid will be highly useful depending on the nature of the bio-fuid tested from patients (e.g., whole blood, serum, or plasma) [36].In the last decade, most studies determined whether LAMP with biosensor could distinguish between nucleic acid that difers by a single base mismatch.
We have previously developed a reverse transcriptionbased LAMP (RT-LAMP) to provide a highly selective and sensitive platform for miRNA [37].In this work, we also used the LAMP reaction for miRNA detection, but reverse transcription process was replaced by ligation reaction to make the method simple and sensitive.Two stem-loop structures DNA (Linker A/B probes) were designed, which were hybridized with miRNA and linked by SplintR Ligase when the target miRNA appeared.Te LAMP reaction was carried out by adding backward inner primer (BIP)/forward inner primer (FIP) and Bst 2.0 DNA polymerase.Compared with other self-assembly amplifcation methods, LAMP greatly improved the amplifcation efciency.Finally, we added the double-stranded DNA fuorescent dye EvaGreen to detect the amplifcation product and achieve the quantifcation of the target miRNA, as shown in Scheme 1. Te selectivity of this method was evaluated by testing miR-200a and other miRNAs from the miR-200 family.Our method can efectively distinguish miR-200a from other homologous family miRNAs with high sequence similarity.Tis method has also been successfully applied to quantitatively detect the amount of miR-200a in cell samples, indicating its clinical value.Terefore, this novel detection method could open a new path for the highly sensitive quantitative analysis of miRNA, thereby bringing convenience to clinical diagnosis and prognostic research.

Material and Apparatus
. miRNA 1st Strand cDNA synthesis kit was purchased from Vazyme (Nanjing, China), and Bst 2.0 DNA polymerase and the corresponding isothermal bufer were purchased from New England Biolabs (USA); dNTPs and RNase-free water were all obtained from Takara (Dalian, China).All nucleic acid in this work including Linker A, Linker B, and inner primer (FIP and BIP) were synthesized and purifed by Sangon Biotech (Shanghai, China).miRNAs were synthesized and purifed by Genepharma (Shanghai, China).Te sequences of all nucleic acids are listed in Table S1.Te real-time fuorescence measurements of the LAMP reactions were proceeded on a StepOne (Applied Biosystems) real-time PCR instrument and had the following sequences.

Procedure of LAMP Assay.
In a typical experiment, 9 μL reaction mixture containing 1 μL target miRNA, 1 μL each Linker A and B probes 1, 25 nM and 1 μL of 0.8 mM dNTPs, 1x mM in SplintR Ligation bufer was heated to 95 °C for 5 min and chilled on ice for 2 min, then 1 μL 7 U of SplintR Ligase was added and incubated at 16 °C for 30 min.Te SplintR mixture solution (10 μL) was added into a total volume of 20 μL in 1x isothermal amplifcation bufer to form LAMP Mix composed of forward inner primer (FIP), backward inner primer (BIP) 80 nM, and 8 U of Bst 2.0 DNA polymerase.1 × EvaGreen dye was added to the reactions following primer annealing but prior to the addition.Te reactions were analyzed using the Step OnePlus real-time PCR machine that was set up to incubate the samples for 60 cycles of two-step incubations: step 1: incubation at 61.5 °C for 150 s and step 2: incubation at 61.5 °C for 30 s (total incubation time of 3 min/cycle unless otherwise indicated).Te resulting data were analyzed using the Step OnePlus analysis software to generate Ct (quantifcation cycle) values for each amplifcation, and Ct was redefned by multiplying Ct by 3 in this paper.

Cell Lysis and RNA Preparation. Te human colon cancer cells (HT29) and human hepatoma cells (BEL7402)
were gifts from Department of Pharmacology, College of Pharmaceutical Sciences, Zhejiang University.Cell lines were maintained according to instructions from the ATCC.HT29 and BEL7402 were collected and centrifuged at 3000 rpm for 5 min in culture medium, washed once with PBS bufer, and then spun down at 3000 rpm for 5 min.Total RNA was extracted from human colon cancer cells using miRNeasy Kit according to the manufacturer's procedures.Te sample of miR-200a in these cells was diluted and then analyzed with the proposed miRNA detection method.

Design Principle of this Method
Based LAMP.Tis miRNA detection strategy contains two linker probes and two primers; both of them are from the typical LAMP reaction.Te LAMP system, which consists of two hairpin 2 International Journal of Analytical Chemistry DNA probes (Linker A and Linker B) are designed and constructed as a dumbbell DNA initiator to get an active amplicon.Te Linker A/B probes have an overhang complementary to the half of target miRNA.Only when it is perfectly hybridized with miRNA, the ligation of these two probes would be carried out in the presence of SplintR Ligase to aford high specifcity in identifying the mutations in target miRNA.Te LAMP process would start with the coming of FIP/BIP primer where intact inner primers bind and extend.Te fuorescent dye (1x EvaGreen) was added to the reactions following primer annealing with the addition of Bst 2.0 DNA polymerase.In the absence of miRNA or there is a mismatch between miRNA, no dumbbell-shaped amplicons structured product is obtained, therefore, precluding the LAMP amplifcation with merely giving a low and a late increase of the fuorescence signal.Tis miRNA-LAMP assay could get a high sensitivity and selectivity according to the real-time fuorescence curve and Ct value which were recorded in LAMP reaction.

Design of Linker A/B Probes.
To explore the feasibility and more investigate of this method in practical use, we designed and compared two templates (MERS 1a and MERS 1b), which came from the original LAMP.Tis two Linker A/B probes extension mechanism of the miRNA-LAMP system were tested for miR-200a detection, which is well a known biomarker overexpressed in the luminal breast cancer (strongly downregulated during oncogenic EMT) [42].Te switch from loop structure is a key factor for the amplifcation mechanism, thus the design of Linker A/B probes is important for the detection.Te real-time fuorescence intensity distribution curves were used to investigate the efciency of Linker A/B probes from two templates.After we tested in parallel with diferent concentrations from 1 nM and 10 pM of target miR-200a, the probes from MERS 1b template showed high sensitivity and changed in the fuorescence intensity function of target concentration by real-time RT-PCR, as shown in (Figure 1).

Amplifcation Reaction Steps for Detection Method.
To achieve a highly sensitive detection of miRNA, we optimized the miRNA-LAMP reaction by investigating different operation strategies.One-step reaction method where we put all the enzyme, target, and primers in one tube and then start the amplifcation directly were compared with two-step method (ligation and amplifcation).Te quality of the Ct value for diferent concentrations of the target are shown in Figures 2(a)-2(c); the two-step method showed better performance in the amplifcation curve and enhanced sensitivity for miRNA detection.

Ligase Enzyme Selection.
To achieve the highestefciency ligation between the Linker A/B probes mediated by ligase enzyme to form the dumbbell-shaped, the approach was optimized by testing three diferent types of ligase enzymes.SplintR Ligase, Taq DNA Ligase, and T4 DNA Ligase were applied miRNA-LAMP detection.As shown in the (Figures 3(a International Journal of Analytical Chemistry the other two enzymes (T4 DNA Ligase and Taq DNA Ligase), which ligated the two adjacent single-stranded DNA splinted fragments with high efciency by a complementary miRNA strand.

Optimization for the miRNA-LAMP Assay.
To establish the detection method and reduce the number of false-positive results, several parameters were systematically investigated for miRNA-LAMP detection.Tese parameters included the concentrations of Linker A/B, FIP and BIP primers, SplintR Ligase, 2.0 DNA polymerase, and the reaction temperature.Te concentration of Linker A/B led to an increase in the ΔCt value, which reached the maximum at 50 nM and then decreased gradually (Figure S1).Te ΔCt value reached the maximum when primers FIP/BIP concentration is 800 nM and then decreased gradually (Figure S2).Te ΔCt value also reached a maximum for a Bst 2.0 DNA polymerase amount of 8 U (Figure S3) and SplintR Ligase amount of 4 U (Figure S4).Furthermore, we investigate the LAMP amplifcation temperature ranging from 59 °C to 65 °C; the ΔCt values for each diferent amplifcation temperature were compared when the amplifcation temperature is 61.5 °C and the results show lower ΔCt values from miRNA (Figure S5).

Selectivity.
To evaluate the selectivity of this miRNA-LAMP method, interference assays were performed under identical conditions using other miRNA from miR-200 family, such as miR-200b, miR-200c, and miR-429, due to the high sequence homology (Table S1).As shown in Figure 4, the Ct value was measured from perfectly complementary targets miR-200a (1 pM) to other miR-200 family miRNAs (10 pM).Te results suggested that high amplifcation and lowest Ct value for miR-200a target compared to other members of the family, which demonstrated that this approach for detection miRNA had high selectivity and sensitivity.Our miRNA assay can efectively discriminate between members of multiple closely related sequences of miRNAs from the miR-200 family.

3.7.
Quantifcation of miRNA-LAMP Assay.Under the optimized conditions, diferent concentrations of miR-200a were analyzed by monitoring the changes in the Ct value.Te realtime fuorescence intensity curves all exhibited a sigmoidal shape, and gradual increases of the Ct values were observed with decreased miR-200a concentrations from 100 fM to 10 nM.A fuorescence signal produced by a miR-200a concentration as low as 100 fM could be clearly discriminated from that of the blank control (Figure 5).Te miR-200a ranged between 100 fM to 5 nM, with the correlation equation Ct � −3.7485l og C + 57.364 (R 2 � 0.9859).Te sensitivity of this assay compared favorably with previous eforts for miRNA detection, as summarized in (Table S2).

Assay of miRNA in Cell
Samples.Te proposed methods were successfully used to quantify the amount of miR-200a in the total RNA sample that was extracted from cells.Human colorectal cancer cells HT29 were used as positive control and human liver cancer cell BEL-7402 as a negative control (low expression miR-200a).We detected the miR-200a level from the total RNA extracted concentration not exceeding 250 ng; the results of miRNA-LAMP assay were in good agreement with those obtained from other stem-loop RT-PCR.miR-200a in two types of cells HT29 and BEL-7402 were detected.Te results indicate that HT29 and BEL-7402 cells contain 73.3 pM and 6 fM of miR-200a in total RNA 415 ng and 316.8 ng.

Conclusion
In this section, a simple and efcient miRNA-LAMP detection method based on development self-assembly amplifcation to LAMP is established.In this method, the smart 6 International Journal of Analytical Chemistry design of the two probes with the loop was specifcally designed to form two diferent stem-loop structures before and after binding to the target miRNA.Te sensitivity of the LAMP method is comparable to that of the self-assembly amplifcation method, but the upper limit of quantifcation of the miRNA-LAMP method is higher and showing a wider range of quantifcation, which is feasible for the accurate of miRNAs down to the 100 fM level in real samples.Moreover, this proposed miRNA-LAMP assay does not require any modifed or labeled DNA probes, and only one type of ligase enzyme with DNA polymerase is needed, which should signifcantly reduce the cost and simplify the experimental procedure.At the same time, the miRNA-LAMP-based miRNA detection method we just need to modify the complementary sequences of the probe and target miRNA can easily be extended to the detection of other small RNAs, including miRNA, so it has universal value.Te versatile miRNA-LAMP miRNA assay not only remarkably simplifes the probe design for efcient LAMP amplifcation but also leads to high sensitivity and specifcity.

Figure 5 :
Figure 5: Fluorescence intensities (a) and Ct values (b) of miRNA-LAMP assay.Experimental conditions: Linker A/B 10 pM, 0.8 mM dNTP, 4 U SplintR Ligase, 46 nM FIP and BIP primers, and 8 U Bst 2.0 DNA polymerase.Detection was performed as described in material and methods.